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Erythrocyte Membrane-Enveloped Polymeric Nanoparticles as Nano-Vaccine for Induction of Antitumor Immunity against Melanoma Yuanyuan Guo 1, Dong Wang 1, Qingle Song 1, Tingting Wu 1, Xiangting Zhuang 1, Yuling Bao1, Miao Kong 1, Yan Qi 1, Songwei Tan 1 and Zhiping Zhang1,2,3,*
AUTHOR ADDRESS 1
Tongji School of Pharmacy
2
National Engineering Research Center for Nanomedicine
3
Hubei Engineering Research Center for Novel Drug Delivery System
AUTHOR INFORMATION Corresponding Author Prof. Dr. Zhiping Zhang Fax and Phone: +86-27-83601832 E-mail:
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ABSTRACT
Cancer immunotherapy is mainly focused on manipulating patient’s own immune system to recognize and destroy cancer cells. Vaccine formulations based on nanotechnology have been developed to target delivery antigens to antigen presenting cells (APCs), especially dendritic cells (DCs) for efficiently induction of antigen–specific T cells response. To enhance DC targeting and antigen presenting efficiency, we developed erythrocyte membrane-enveloped poly (D,L-lactide-co-glycolide) (PLGA) nanoparticles for antigenic peptide (hgp10025-33) and toll like receptor 4 agonist, monophosphoryl lipid (MPLA). Mannose-inserted membrane structure was constructed to actively target APCs in lymphatic organ and redox-sensitive peptide-conjugated PLGA nanoparticles were fabricated which prone to cleave in the intracellular milieu. The nanovaccine demonstrated the retained protein content in erythrocyte and enhanced in vitro cell uptake. Antigen-depot effect was observed in administration site with promoted retention in draining LN. Compared with other formulations after intradermal injection, the nano-vaccine prolonged tumor-occurring time, inhibited tumor growth and suppressed tumor metastasis in prophylactic, therapeutic and metastatic melanoma models, respectively. Additionally, we revealed that nano-vaccine effectively enhanced IFN-γ secretion and CD8+ T cell response. Taken together, these results demonstrated the great potential in applying erythrocyte membraneenveloped polymeric nano-platform for antigen delivery system in cancer immunotherapy.
KEYWORDS cancer vaccine· red blood cell· Nanoparticles · dendritic cells·immunotherapy
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The current cancer immunotherapies, such as cytokines treatment, immune checkpoint blockade immunotherapy, adoptive immunotherapy and chimeric antigen receptor T cell Immunotherapy (CAR-T), have shown meaningful benefits for cancer therapy.1 Among of them, dendritic cells (DC)-based immunotherapy plays crucial role in cancer treatment as the unique features of DC in the immune system. As the most potent professional antigen presenting cells (APCs), DC is considered as the important initiator of immunity for generation of specific cytotoxic T lymphocytes (CTL)-mediated immunotherapy against tumors. To promote T cell activation, DC has to migrate to the proximal draining lymph nodes (LN) after capture of tumor antigens, and secrete the pro-inflammatory cytokines subsequently.2 Provenge®, the first FDA-approved therapeutic cancer vaccine, is based on the ex vivo pulsing of autologous DCs with prostatic acid phosphatase, an antigen associated with a subset of prostate cancers.3 Despite DC-based immunotherapy presents the induction of antigen-specific responses in preclinical animal models and human clinical studies, the isolation, culture and antigen-pulsing of DC is still at high cost, extremely labor-intensive, and less reproducible. Moreover, transplanted DC which could home to the LN was only 0.5-2.0% and thus not enough to induce efficient T cell responses.4, 5 To avoid the tedious ex vivo DC-based procedure and present sufficient amount of antigen to DC for efficient induction of antitumor CTL responses, nanotechnology is applied for sustained and targeted antigen delivery to DC.6-8 In this regard, nanoparticles (NPs) of natural or synthetic origin have been developed to enhance the efficacy of therapeutic agents for antigen delivery.7, 9 Soluble antigens are encapsulated into nanocarriers to enhance uptake by professional APCs with less proteolytic degradation and improved stability.10, 11 Furthermore, with co-encapsulation of adjuvant, antigen-loaded NPs can induce in situ DC maturation and much stronger T cell
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responses. To date, the antigen delivery system mediated by nanocarriers have exhibited great potential in cancer immunotherapy.6, 8, 10, 11 Many kinds of NP delivery system have been developed for cancer chemotherapy and immunotherapy, including polymeric NPs, microspheres, liposomes, cell or cell-derived vesicles, etc.8, 11-13 Among of them, polymeric NPs, especially applying FDA approved polymers (e.g. poly (D,L-lactici-co-glycolic acid), PLGA) as delivery system have attracted much attention.6, 8, 14
In our previous research, we have reported PLGA NPs as melanoma-associated antigenic
peptides, TRP2180-182 and hgp10025-33, delivery system. TRP2 peptide and toll-like receptor 4 agonist (MPLA) co-entrapped PLGA NPs are capable of promoting presentation of antigen in APCs.8 Recently, cell or cell-derived membrane vesicles have been proposed as potential drug delivery carriers, including erythrocytes (RBCs), macrophages, dendritic cells, stem cells, tumor cells and so on.13, 15-18 Membrane vesicle-based drug delivery has received particular attention by virtue of their biological formation, antigenic components and physicochemical properties.19 For example, tumor cell-derived microparticles can be used as a good package of chemotherapeutic drugs, which exhibited anticancer activity with reduced adverse effects.13 Compared with the above mentioned cultured cell-derived vesicles, red blood cell (erythrocyte, RBC) may be more appropriate for selective delivery of pharmacological agents as its convenient isolation, intrinsic biocompatibility, and a variety of other remarkable properties.15 Erythrocyte is an interesting tool for antigen delivery to target DC and induce CTL responses.20-23 As an antigen carrier, RBC can protect the antigens from blood clearance, deliver antigens in strategic organs and present antigens directly to immune cells after processing by APCs.23, 24 The ovalbumin antigen entrapped-RBC can promote the phagocytosis by APCs and result in the subsequent activation and proliferation of antigen-specific CD4+ and CD8+ effector T cells.22 HIV regulatory protein,
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TAT conjugated on the RBC membrane exhibited a 1250-fold less dosage to induce a similar Tcell response compared with soluble TAT.25 Several distinct methods have been developed to load antigen in RBCs or onto the outer surfaces by physical encapsulation or chemical conjugation. The amount of antigen or drug entrapped in RBC is variable and the encapsulated substances leak rapidly from the RBC by the mainly used osmosis-based method.23, 25 The electroporation and biotin-avidin-biotin bridge may cause the disruption of cell membrane and affect the functionalities of proteins in RBC.26 Recently, functionalizing synthetic NPs with natural RBC membrane have been developed to construct RBC-mimicking NPs. The RBC membrane-cloaked NPs have been developed as a carrier to extend drug circulation time in cancer chemotherapy and a safe vaccine to deliver pore-forming toxins to immune systems.27,28 With this in mind, an alternative approach is to directly employ RBC membranes as building materials to construct antigen carriers. In the present work, we proposed a novel antigenic peptide delivery system with erythrocyte membrane-enveloped PLGA NPs. The nanocarriers will combine the loading capacity of PLGA NPs and the natural intrinsic properties, easy modification with targeting ligand and insertion capacity for lipid-like adjuvant of MPLA in the membrane structure of RBC. The feasibility of polymeric NPs as peptide and adjuvant delivery has been reported to efficiently induce functional CTL specific to tumor-associated antigens.7 Compared to the hydrophobic melanomaassociated antigenic peptide TRP2180-182, hgp10025-33 with high hydrophilicity can hardly be entrapped in PLGA NPs by double emulsification method with entrapment as low as 0.5%, which limited its application in vaccine formulations.8 The conjugation of cysteine-modified peptide with polymer via disulfide bond may be an alternative solution with enhanced entrapment efficiency and prone to cleave under the reductive condition of abundant γ-glutamyl-
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cysteinyl-glycine (GSH) in the intracellular milieu.29, 30 The bio-reducible NP has been shown to enhance vaccine-induced antibody production and CD8+ T cell-mediated tumor cell lysis.31 Therefore, PLGA-SS-hgp NPs (PLGA-NPhgp) were fabricated with reductive-cleaved hgp100 (hgp) conjugation and the surface of NPs was further decorated with RBC membrane (Figure 1). The mannose receptor (MR) is a carbohydrate-binding receptor expressed on macrophages, DCs and nonvascular endothelium.32 More importantly, it is a key molecule in antigen recognition.33 The activation of MR can arouse the targeting of APCs. Mannose-modified NPs enhanced vaccine delivery into draining LNs and increased vaccine-induced anti-tumor immune responses.34 DSPE-PEG-Mannose (DSPE-PEG-Man) was thus incorporated into RBC membrane to generate DSPE-PEG-Man-inserted-RBC (Man-RBC), which presumably would be able to actively target APCs in lymphatic organs. Here, we characterized PLGA-SS-hgp@RBC NP (RBC-NPhgp) and PLGA-SS-hgp@Man-RBC NP (Man-RBC-NPhgp) in vitro and in vivo to evaluate the nano-vaccine formulations on melanoma immunotherapy. RESULTS AND DISCUSSION Preparation and Characterization of RBC membrane-enveloped PLGA NP (RBC-NP) PLGA-SS-hgp was synthesized via a three-step reaction (Supporting Information, Figure S1). After activation of carboxylic acid terminated PLGA with NHS, 2-(pyridyldithio)-ethylamine (PDA) was introduced to synthesize PLGA-PDA intermediate. The pyridyl sulfide group of PLGA-PDA was then exchanged by cysteine-modified hgp peptide, hgp-SH (KVPRNQDWLC) to form PLGA-SS-hgp. The conjugation of hgp to PLGA was confirmed by 1H-NMR (Supporting Information, Figure S2). Four methyl protons of valine and leucine were used as characteristic peaks at 0.79-0.94 ppm. Protons of some peptide bonds and aromatic ring of tryptophan appeared at 6.80-8.50 ppm, while the peaks belonging to PDA disappeared at the
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same area. The response of PLGA-SS-hgp in reductive environment was evaluated under the condition of DL-Dithiothreitol (DTT) and the hgp content was calculated by HPLC measurement. No retention peak of hgp was observed in the sample of PLGA-SS-hgp without DTT treatment in contrast to free hgp-SH or DTT treated sample (Supporting Information, Figure S3). These results confirmed the successful synthesis of PLGA-SS-hgp and reductivecleavage behavior of integrity peptide under in vitro reductive environment. RBC-NP was fabricated according to preparing the PLGA NP by nanoprecipitation, deriving RBC membrane vesicles from natural RBCs by hypotonic hemolysis and coating the RBCmembrane onto the surface of PLGA NP by serial extrusion.14, 35 In order to visualize the structure of RBC-NP, the particles were negatively stained with phosphotungstic acid and observed through transmission electron microscopy (TEM) (Figure 2a and b). A spherical coreshell structure was exhibited as expected in RBC-NP (Figure 2b). Moreover, the average hydrodynamic diameter of RBC-NP was increased from 131.3±0.6 nm for PLGA-NP to 149.2±0.6 nm, with slightly increase of 17.9 nm (Figure 2c). The shell-structure and thickness detected in TEM images and increased particle size measured in dynamic light scattering (DLS) were in good agreement with the thickness of bilayer structure of natural RBC membrane which is known to be 5-10 nm.36 Man-RBC-NP, RBC-NPhgp and Man-RBC-NPhgp were prepared in the similar way. Physiological stability is a significant challenge in the application of NP. Designed for biomedical applications, we evaluated the stability of PLGA-NP and RBC-NP by suspending them in two commonly used biological media, pH 7.4 phosphate buffer solution (PBS) and pure fetal bovine serum (FBS). The particle size and the surface zeta potential were continuously monitored by DLS for 15 days in PBS. The hydrodynamic diameter of RBC-NP was slightly
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changed from 149.2±0.6 nm to 156.6±4.6 nm, the zeta potential decreased from -17.5±2.0 mV to -20.1±0.7 mV (Figure 1d). It seems that no significant change was observed in PBS for at least 15 days. For the serum stability, we shelved the RBC-NP for 4 h in FBS to monitor the absorbance change at 560 nm.35 Cross-linking between NP would be expected to happen if serum proteins bind to the surface of NP.37 It is well-known that larger particles induce higher light scattering. Aggregation of unstable particles can thus be assessed by monitoring the increase in absorbance value.35 The results suggested that RBC-NP was more stable than PLGA NP in serum as none observable changes in absorbance within 4 h (Supporting Information, Figure S4). It may be attributed to that the RBC membrane-covered structure prevented the serum binding from RBC-NP. An important feature of the bio-reducible NPs is prone to cleavage in the presence of reductive agent, such as DTT, and then quickly releasing their cargoes. Accordingly, we evaluated the in vitro release profile of PLGA NPhgp and RBC-NPhgp by co-incubation with 10 mM DTT. As shown in Figure 2e, the PLGA NPhgp and RBC-NPhgp showed controlled release behavior over 48 h, while negligible hgp release was detected in the absence of DTT. Moreover, RBC-NPhgp exhibited slower sustained release of hgp compared with PLGA NPhgp in the presence of DTT which may be caused by the RBC barrier from this membrane-coated NP. It demonstrated that the bio-reducible bond conjugated peptide-loaded NPs can realize preferentially intracellular stimuli-responsive release property instead of a burst release before uptaken by DCs. To ensure the retained membrane proteins of RBC in RBC-NP after extrusion, SDS-PAGE of Man-RBC-NP along with PLGA NP, natural RBC membranes, Man-RBC, and RBC-NP were performed in parallel analysis (Supporting Information, Figure S5a). Compared with natural RBC membranes, the majority of protein content did not lost during the samples preparation. The
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protein profiles of Man-RBC-NP and RBC-NP were as similar as natural RBC membranes. To confirm the presence of specific antigens on the RBC-NP, CD58 and CD59 were examined by Western blotting analysis, respectively. The results showed that CD58 and CD59 molecules both exsited no matter pre-/post- membrane extrusion (Supporting Information, Figure S5b and S5c). CD59 is a small (20 kDa), globular, glycophosphatidyl inositol (GPI) linked glycoprotein on almost all tissues in the body and all circulating cells.38 CD58 is a highly glycosylated cell adhesion molecule (55-70 kDa) that is expressed in diverse cell types as a transmembrane or GPI-membrane-anchored form.39 CD58 and CD59 that distributed on the RBC surface are the corresponding ligands of CD2.40 CD2 presents on the surface of T lymphocytes that is a member of the immunoglobulin superfamily and plays a crucial role in cell-cell adherence.41 CD58 and CD59 are considered to play an important role in the interaction of T cells with target cells. Taken together, these observations suggested that RBC-NP was stable enough to apply in biomedical conditions and the majority of membrane proteins were retained in RBC-NP throughout the particle preparation process. Man-RBC-NP enhance cell uptake and promote retention in draining LN A key step by which naive T cells first become aware of tumor-associated antigens is the presentation of tumor antigens by host APCs. NP (20-200 nm) is known to traffic to the draining LN rapidly after intradermal injection where they are effectively taken up by resident DCs.42 We supposed that the Man-RBC-NP may efficiently home to the LN when intradermally injected, because the NP was small enough (
